This invention relates generally to mass spectrometry, in particular to a novel apparatus and method to prepare an ion pulse for ideal analysis in a time-of-flight mass spectrometer and in tandem mass spectrometers in which fragments are analyzed via time-of-flight mass spectrometry.
Mass spectrometers are devices which vaporize and ionize a sample and then determine the mass to charge ratios of the collection of ions formed. One well known mass analyzer is the time-of-flight mass spectrometer (TOFMS), in which the mass to charge ratio of an ion is determined by the amount of time required for that ion to be transmitted, under the influence of pulsed electric fields, from the ion source to a detector. TOFMS has become widely accepted in the field of mass spectrometry, having the desirable attributes of high scan speed, high sensitivity, theoretically unlimited mass range, and, if an ion mirror is used, achievable resolutions of greater than 10,000. The spectral quality in TOFMS reflects the initial conditions of the ion beam prior to acceleration into a field free drift region. Specifically, any factor which results in ions of the same mass having different kinetic energies, and/or being accelerated from different points in space, will result in a degradation of spectral resolution, and thereby, a loss of mass accuracy. High mass accuracy is a desirable property in spectrometers used in the analysis of biomolecules, as it is one of the important factors in the unambiguous determination of peptide, and thereby protein, identity using database searching.
Two instrumental developments which minimize the effects of spatial and energy spreads on the final spectra are prevalent in the field. The first is the two-stage, or Wiley-McLaren, acceleration source, which provides first order space focusing, and the second is the ion mirror, or reflectron, which provides first order energy focusing. Additionally, the two widely adopted methods to produce gas phase biomolecular ions for mass spectrometric analysis, namely matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), have integrated certain instrumental attributes which have enhanced spectral resolution. The development of delayed extraction (DE) for MALDI-TOF as described in U.S. Pat. Nos. 5,625,184, 5,627,369 and 5,760,393 has made high resolution routine for MALDI based instruments. For ESI-TOFMS, high resolutions have been achieved by transmitting the ion beam through an RF only quadrupole and into the acceleration region of a TOFMS. The accelerating pulse is applied perpendicular to the direction of transmission. For both these methods, however, the resolution enhancement is not achieved without sacrificing another element of instrumental performance.
In DE-MALDI, a short delay is added between the ionization event, triggered by the laser, and the application of the accelerating pulse to the TOF source region. The fast (i.e., high-energy) ions will travel farther than the slow ions, in effect transforming the energy distribution upon ionization to a spatial distribution upon acceleration. A Wiley-McLaren source is used for space focusing. The delay time in DE-MALDI, however, can only optimize performance across a narrow range of mass to charge ratios, hence, resolution varies across the spectrum and calibration is non-linear. Additionally, the performance of the spectrometer is strongly coupled to the energy distribution from the ionization source. The highest mass resolution is achieved using so-called xe2x80x9cthresholdxe2x80x9d conditions, i.e., operating the laser at the minimal fluence that yields observable ionization. If laser fluence is increased beyond this threshold value, ions are formed with a broader energy distribution, thereby degrading spectral quality.
It is known in the art that raising the laser fluence substantially above the threshold value increases the number of ions formed per laser pulse by orders of magnitude. As a consequence, in DE-MALDI the resultant direct coupling of the ionization source with the spectrometer is manifested in a tradeoff between resolution and sensitivity, that is one cannot simultaneously optimize conditions for ionization and mass analysis. An independent problem in MALDI based spectrometers is the observation, in some instances, of spectral features resultant from decay of ions during their flight time from the acceleration source region to the detector. Briefly, if ions created in the MALDI process are formed with excess internal energy, ions may dissociate prior to detection. The resulting fragments appear in the spectrum as unassignable chemical noise, xe2x80x9cmetastablexe2x80x9d peaks, and/or increased background in the spectrum.
In a spectrometer equipped with an ESI source, a method termed orthogonal acceleration (oa) TOFMS is typically used. In oa-TOFMS, the ionization source may be separated from the acceleration region of the TOFMS by an RF-only quadrupole operating in the millitorr pressure regime. This quadrupole acts as a beam guide transmitting ions formed at atmosphere into the vacuum regions of the spectrometer. As described in U.S. Pat. No. 4,963,736, the passage of an ion beam through an RF-only quadrupole operated in the millitorr pressure regime leads to the xe2x80x9ccollisional coolingxe2x80x9d of the beam. Through sequential collisions between the ion and the background gas, the internal energy of the ions is lowered to approach that of the background gas (i.e., the ion beam becomes thermalized). Similarly, the translational kinetic energy of the beam is lowered, restricting the motion of the ions to the low field region of the quadrupolar potential, resulting in a narrow beam of ions and more efficient transmission through restrictive ion optics. Lastly, reduction of the translational kinetic energy of ions coaxial to the beam, results in a denser beam with a smaller translational energy spread. As collisional cooling lowers the internal energy of the ions formed, harsh ionization conditions can be used without degrading spectral resolution and thus in an oa-TOFMS the ionization source becomes effectively decoupled from the spectrometer. The oa-TOFMS has been coupled to a MALDI ionization source, operated with a high repetition rate, high fluence Nd:YAG laser OPO 5000, as described by Anatoli Verentchikov et al. xe2x80x9cCollisional Cooling and Ion Formation at Intermediate Gas Pressurexe2x80x9d, Proc. 47th ASMS Conference on Mass Spectrometry and Allied Topics, 1999, to create a quasi continuous beam which is pulsed into the TOFMS.
A key element of oa-TOFMS is that the beam enters the acceleration region of the TOFMS orthogonal to the direction the pulse is accelerated. (see U.S. Pat. No. 5,117,107 and Dodonov USSR Patent No. SU 168134A1 and published PCT application WO91/03071). Thus, the initial conditions of the accelerated TOF pulse are defined by the properties of collisional cooling in a quadrupolar potential, i.e., the ions have small spatial and energy distribution. One limitation in oa-TOFMS is that the duty cycle of the instrument, which is defined as the ratio of the time required to fill the acceleration region of the TOF spectrometer to the time for mass analysis, is typically a low 5-20%. A further disadvantage of oa-TOFMS is that the ions of the accelerated pulse maintain a small velocity component in the direction perpendicular to TOF acceleration. Therefore the ion pulse accelerated in the TOF has a natural xe2x80x9cdriftxe2x80x9d angle which must be compensated for, either through the use of a large detector surface or an electrostatic steering deflector, a device which is known in the art to degrade resolution.
The problem of poor duty-cycle in oa-TOFMS has been addressed in a combination, or xe2x80x9chybridxe2x80x9d instrument in which the continuous ion beam is stored in a quadrupole ion trap and ejected as discrete pulses into the TOFMS by Mark Q. Qian et al. xe2x80x9cProcedures for Tandem Mass Spectrometry on an Ion Trap/Reflection Time-of-Flight Mass Spectrometerxe2x80x9d, Rapid Communications in Mass Spectrometry, 10, 1996. According to the authors, careful synchronization of the emptying of the trap and the TOF analysis can be used to achieve a near 100% instrumental duty cycle. With few exceptions, these systems use a commercial ion trap with a conventional geometry for both storage and creation of the TOFMS acceleration field electrodes. The use of the trap geometry for ion extraction is problematic, as the trap electrodes create a non-linear electric field, while optimal TOFMS operation, requires a linear electric field. Two references, U.S. Pat. No. 5,569,917 and published PCT Application WO 99/39368, discuss novel combinations of extraction voltages that can be used in conjunction with the conventional ion trap geometry to create and improve ion pulses for TOFMS. In each case, however, reference is made to using differential extraction voltages to compensate for higher order fields in the trap itself and neither reference demonstrates the resolution of either oa-TOFMS or DE-TOFMS systems. Existing work on MALDI-trap TOFMS, as described by Peter Kofel et al. xe2x80x9cMatrix Assisted Laser Desorption/Ionization Using a New Tandem Quadrupole Ion Storage Trap, Time-of-Flight Mass Spectrometerxe2x80x9d, Rapid Communications in Mass Spectrometry, 19, 1996, has demonstrated that as the electric field in the center of the quadrupolar potential is substantially linear, ions are sufficiently collisionally cooled.
The issue of a poor extraction field in ion trap TOFMS systems has been addressed by the use of a segmented ring ion trap (see, for example, Qinchung Ji et al. xe2x80x9cA Segmented Ring, Cylindrical Ion Trap Source for Time-of-Flight Mass Spectrometryxe2x80x9d, Journal of the American Society of Mass Spectrometry, 7, 1996) the purpose of which was to couple an electron impact source to a TOFMS with a 100% duty cycle. In this instrument the xe2x80x9ctrapxe2x80x9d was created by four simple ring electrodes, operated such that an oscillating field which is substantially quadrupolar was created. Ions are trapped in the field formed by the rings for a set period of time. At the end of the trapping period, the RF potential is rapidly switched off and a unidirectional, linear field in the (former) trapping volume is actualized by applying DC pulses to the rings, the magnitudes of which are proportional to the distance from that electrode to the source plate. Resolution attained on this TOFMS, however, was not optimal. Although an ideal extraction field was claimed to be formed, the position and energy of the ions at the time the field was applied was found to be strongly dependent on the phase of the quadrupolar potential at the instant the RF power supply was switched off. Ions that are moving in the direction opposite to that of the TOFMS accelerating field required a xe2x80x9cturn aroundxe2x80x9d time during extraction and this additional time degraded the spectral resolution. Also, the phase dependent spread in kinetic energies resulted in the necessity to use a reflectron that was specially designed to accommodate ions with a large velocity spread.
The performance of existing hybrid ion trap-TOFMS instruments has been substantially limited by the following factors:
Initial trapping of the ion beam is inefficient due to the necessity to overcome the barrier created by the rapidly oscillating quadrupolar potential. A significant portion of ions formed will be lost in the injection process unless the ions are formed within the trapping volume.
When a continuous ion beam is used, only those ions that have, through collisions with the background gas, lost sufficient translational kinetic energy to be confined in the quadrupolar potential will have stable trajectories in the ion trap. Consequently trapping efficiency is low.
The conventional electrode geometry of the three-dimensional ion trap has a relatively low space charge capacity. If, for example, more than 1000 ions are confined in 1 mm3, energy gained from inter-ion repulsion will result in the ions having a translational kinetic energy, which is greater than thermal energy, thereby lowering TOF resolution. For typical trap operating conditions of 1 millitorr of helium, full collisional cooling requires approximately 30 ms. Thus, to maintain ions at thermal energies, total throughput of the system must be below 3xc3x97104 ions per second, a value which is not adequate for most applications.
In order for ions to be stored effectively, typically 1 millitorr of helium is present in the trap. However, since the same volume is used for storage and acceleration, during acceleration ions may undergo numerous collisions which alters the ideal trajectories in the TOF analyzer. Additionally, in the three-dimensional ion trap, the combination of poor confinement of the ion beam and the non-linear acceleration field result in a wide ion cloud to extract; therefore, to enhance sensitivity a large extraction aperture is used between the trap and TOFMS. This raises the pressure in the flight tube and thereby increases both the number of collisions which transpire and the load on the vacuum pumps in the flight tube.
Another broad application of mass spectrometry is tandem mass spectrometry, denoted MS/MS. An MS/MS instrument provides the capability to isolate an ion based on its mass to charge ratio, fragment the selected ion, and mass analyze the fragments. Spectra from MS/MS instruments are used to provide information on the structure and bond strength of the precursor ions (sometimes called parent ions). Additionally, through reducing the amount of chemical noise, MS/MS machines actually improve the spectral signal to noise ratio and hence the detection limit of the precursor ions.
The ability of TOFMS to provide parallel analysis of all mass components is used in multiple tandem instruments, classified as hybrid TOFMS. The most common of these hybrid instruments combines quadrupole and TOF technology, often referred to as QqTOFMS. An example of a QqTOFMS has been described by Howard R. Morris et al. xe2x80x9cHigh Sensitivity Collisionally-activated Time-of-Flight Mass Spectrometerxe2x80x9d, Rapid Communications in Mass Spectrometry, 10, 1996. This instrument is constructed from two tandem quadrupoles and an orthogonally situated TOFMS. The first quadrupole, a mass filter, is used for precursor ion selection; fragmentation is precipitated via sequential low energy collisions with an inert gas in an RF-only quadrupole operating in the millitorr regime. The resultant fragment ions are analyzed by an oa-TOFMS. Increasing precursor selection resolution in the mass filter results in decreasing sensitivity, thus achievement of unit resolution is only possible with significant ion losses. Consequently, resolution is compromised in most analytical applications, and the above discussed problems of the second oa-TOFMS, namely poor duty cycle and a drift velocity orthogonal to the TOF axis, also affect performance.
The ion trap TOFMS, can also be operated as a hybrid tandem TOF instrument. In MS/MS mode, during storage precursor ions are isolated and fragmented in a quadrupole ion trap and the contents are analyzed by TOFMS. As the processes of ion isolation and fragmentation are based upon the principles of resonant excitation, the ion traps in such instruments must provide well defined, and near ideal, quadrupolar electric fields. Thus the conventional three-dimensional ion trap electrode geometry operated with a background pressure of 1 millitorr helium is required. The disadvantages of this configuration for TOF analysis were discussed above.
In another hybrid TOF instrument used for MS/MS analysis, the three-dimensional ion trap is replaced with a linear, or two-dimensional, ion trap, (orthogonal to the direction of TOF acceleration) as detailed in published PCT Applications WO 99/30350 and WO 98/06481 and demonstrated by J. M. Campbell et al., as reported in xe2x80x9cA New Linear Ion Trap Time-of-Flight System with Tandem Mass Spectrometry Capabilitiesxe2x80x9d, Rapid Communications in Mass Spectrometry, 12, 1998. In the linear ion trap, ions are confined by a quadrupolar potential in two dimensions and by electrostatic potentials in the third dimension. Thus electrostatic, rather than oscillating quadrupolar, potentials control the flow of ions into and out of the trap and the processes of injection and extraction are both simpler to implement and more efficient than in the three dimensional ion trap. Additionally, the linear ion trap provides a larger trapping volume and thus an enhanced ion storage capacity over the three-dimensional trap. In the above PCT applications, ions were injected into the TOF through coupling lenses. In U.S. Pat. No. 5,763,878, the concept of extracting ions from the linear ion trap through a gap in the rod structure is described, and reference is made to the advantage such a concept would provide for TOF analysis in an oa-TOFMS system. However, the instrument described in this patent suffers from a slow cycle of ion selection and fragmentation in the first MS stage as well as the problems discussed above for all oa-TOFMS.
Another method of TOF based MS/MS analysis uses TOF mass analyzers for both precursor ion selection and fragment ion analysis. U.S. Pat. No. 5,206,508 discusses a TOF/TOF system without a mechanism for precursor ion isolation. A second patent, U.S. Pat. No. 5,202,563, discloses a TOF/TOF system with two reflecting-type mass analyzers coupled via a fragmentation chamber. Lastly, co-pending U.S. patent application Ser. No. 09/233,703, commonly assigned as with the present application, describes a TOF/TOF system and includes a detailed description of a timed ion selector (TIS) used to attain high resolution ion selection with a TOF based system. An instrument based on this patent has been used to record fragment spectra on a wide selection of ions, including biomolecules. This TOF/TOF system has been named a double DE system. Ions are formed in a region with a DE-MALDI source, the precursor ions are selected by the timed ion selector and transmitted to the collision cell. The resultant collection of precursor and fragment ions is transmitted into a second TOF acceleration region. At the time that the ions of interest are near the center of the second source, a high voltage pulse is applied, and the ions are accelerated toward the detector. Varying the time of application of the second acceleration pulse creates the second nominal DE system, through which the resolution of the fragment ion spectra can be optimized.
Various effects limit attainable performance of TOF/TOF instruments (mass accuracy and resolution). Analogous to DE-MALDL the energies and positions of fragment ions entering the second source are dependent on mass to charge ratios. As the velocities of ions entering the second acceleration region of a TOF/TOF spectrometer are orders of magnitude greater than those extracted from a matrix in a standard DE source, limitations known in the art for DE-MALDI (e.g., non-linear calibration) are magnified in the TOF/TOF instrument. Consequently, uniform-focusing conditions cannot be attained across the entire mass range, limiting high resolution (and mass accuracy) to a narrow window of fragment ion masses. In addition, optimization of the resolution in the second MS is strongly dependent on conditions in the first MS, which complicates tuning of the instrument. Furthermore, ions which gain internal energy through collisions, but for which the kinetics of dissociation are such that fragments form during transmission in the field free region of the second TOF, appear as metastable ions in the spectrum, resulting in chemical noise and unassignable spectral features.
In spite of the numerous efforts in the past as reflected by the development of various instruments outlined above, there still is not an apparatus and method that simultaneously addresses all of the ideal requirements of TOF and tandem TOF analyses. For example, the need still exists for an MS instrument wherein final spectral quality is decoupled from the mechanism of ionization, such that conditions that provide maximum instrumental sensitivity (e.g., high laser fluence) can be used without sacrificing spectral quality. Furthermore, if harsh ionization conditions are used, a technique for xe2x80x9ccoolingxe2x80x9d ions that are typically formed with sufficient internal energy to fragment, may be needed within the ion source, such that the spectral degrading effects of metastable fragmentation are suppressed. Ideally, resolution and accuracy should be uniform across the mass range and mass calibration should be linear. Lastly, a 100% duty cycle should be achieved with both pulsed and continuous ionization sources. In addition to the aforementioned desired features, MS/MS analysis using tandem TOF instruments ideally should possess the ability to decouple operation of both the first TOF and second TOF MS stages.
The present inventors have realized that the combined use of dynamic trapping and collisional cooling in a segmented ion trap operating at appropriate gas pressure provides a simple and effective method to prepare an ideal pulse for TOF analysis. In doing so, this invention addresses issues such as instability of ions, poor initial conditions, dependence on laser energy and /or ion losses at the time of ion pulse formation, which heretofore have been a significant limitation of TOFMS. Additionally, the invention addresses problems with respect to the issues of injection into and extraction from an ion storage volume to a time-of-flight mass analyzer. The present invention exhibits a high degree of flexibility and can be implemented in numerous existing TOF systems with MALDI and ESI ion sources, and can be used to substantially improve existing TOF/TOF systems. The invention is also adaptable to various hybrid systems with TOF as a final mass analyzer.
In a preferred embodiment, the invention includes a pulsed ion source (MALDI source or ESI source with a storing and pulsing multipole ion guide), a segmented ion trap filled with gas at about millitorr pressure, and a TOF analyzer. Ions from the source are injected into and dynamically trapped in the ion trap, collisionally confined to the center of the trap and subsequently extracted as a pulse into the TOF analyzer.
Briefly, one preferred embodiment of the invention, as implemented in a single stage TOFMS, operates as follows:
(1) Stable ions are formed using a known ionization mechanism, such as MALDI, ESI, thermospray, ICP, FAB, APCI, etc. sources, that are either pulsed or continuous in nature.
(2) The ions are pulse injected into a segmented ring trap. If MALDI is used, the ionization source could be located external to the trap in a region operated at a higher pressure than the trap. If ESI is used, the ions can be stored in an external ion guide, and pulsed into the segmented ring ion trap.
(3) The ions are trapped via dynamic trapping. The ions are initially confined in the segmented ring trap by rapidly switching on or ramping up a high voltage RF power supply. The applied RF potential creates a quadrupolar field confining the ions in two or three dimensions. In the instance of two-dimensional quadrupolar trapping, the ions are confined in the third dimension through electrostatic potentials.
(4) The ions are velocity damped via collisions with a neutral gas. The subsequent lowering of the ion translational energy will confine ions to the low field (i.e., center) region of the quadrupolar potential.
(5) The ions are pulse extracted from the segmented ring trap and into a TOFMS. This process is accomplished by rapidly switching off the RF potential, and rapidly (e.g., within xcx9c100 ns) applying an extraction potential to the ring electrodes of the trap. The extraction potential is linear and unidirectional, applying to each ring a pulse, the magnitude of which is proportional to the distance from that ring to the first ring electrode.
(6) A pulsed, high voltage, acceleration stage is adjacent to the trapping electrodes, and is differentially evacuated to operate at a pressure intermediate from that of the trap and the TOF flight tube.
(7) The extracted ions are analyzed via the TOFMS. To attain optimal resolution the TOF analyzer is equipped with an ion mirror.
One of the key elements of the invention is a use of a segmented ion trap. Unlike conventional ion traps with hyperbolic-shaped electrodes, a segmented ion trap utilizes multiple planar electrodes. When appropriate RF potentials are applied to these planar electrodes, an approximate quadrupolar field is generated resulting in confinement of ions. During ion extraction, the RF field is turned off and a unidirectional, linear field is achieved through application of suitable DC potentials to the planar electrodes. The invention utilizes two types of segmented trap: a three-dimensional trap, formed by ring electrodes and a two-dimensional trap, also termed xe2x80x98linear segmented trapxe2x80x99, formed by parallel flat plates. Both types of segmented trap are applicable for all the examples discussed below, and the specific type used is selected based on technical conveniences.
The segmented ion trap is used for trapping, storing, cooling and pulsed ejection, but not employed for isolation, excitation, and/or mass analysis. Consequently, there is no need to establish and maintain well defined ion trajectories in the quadrupolar field in the trap. The parameters of the system embodied by the invention can thus be optimized for pulse preparation for TOFMS. In doing so, various aspects of the invention provide numerous advantages and overcome the following problems of the known trap-TOF systems:
Inefficient collisional trapping of a continuous ion beam is replaced by a dynamic trapping of a pulsed ion beam.
Stabilization of ions can be improved when desired by lowering internal energy in gas collisions in the ion source. Gas collisions also lower kinetic energy of ions and thus improve efficiency of dynamic trapping in the segmented trap.
Confinement of ions in the trap can be improved by the use of a smaller size trap and the selection of a stronger RF field at a higher frequency, which allows a broad mass range of ions to be stable in RF field. The optimization becomes possible since the trap is used exclusively for storage and there are no requirements to select and control RF frequencies to maintain precise ion trajectories as imposed by resonant excitation techniques.
For certain applications, the space charge limitation can be reduced by the use of a two dimensional trap, low mass cut off in the trap, and a higher repetition rate of pulsed extraction.
The gas load on the TOF system can be reduced by using pulsed gas introduction into the trap or into the ion source and by the introduction of an additional differentially pumped acceleration stage.
The quality of TOF spectra (resolution and mass accuracy) can be improved by the better confinement of the ion beam, the absence of beam defocusing in a uniform accelerating field, and a low probability of gas collisions during acceleration and within a TOF flight tube.
One preferred embodiment provides a system with collisional stabilization of MALDI generated ions at an intermediate gas pressure with a subsequent pulsed injection into the next differentially pumped stage where ions are dynamically trapped in a segmented trap, wherein the ions are stabilized, confined, and pulse ejected into the TOF. In one particular implementation, the trap is a two dimensional segmented trap and pumping of the analyzer is improved by an additional pumping stage between the trap and the TOF. Both axial and orthogonal coupling geometries with the TOFMS are viable options for this embodiment. Collisional cooling in the source (i.e., prior to the confinement and acceleration region) allows the use of a high repetition and/or high energy laser to enhance sensitivity of analysis. Analyzer performance is decoupled from source conditions, resulting in improved, uniform resolution and a linear calibration.
In one embodiment of the invention, the gas is introduced into the source region via a pulsed valve to reduce gas load on vacuum pumps and to provide a lower gas pressure for ion ejection. In another embodiment of the invention, the gas is similarly introduced into the trap via a pulsed valve and ions are formed in the same differentially pumped stage. In yet another embodiment of the invention, an infrared laser is used to produce initially stable ions and gas pressure is reduced to the minimum sufficient for ions confinement. It is known in the art that use of an infrared laser with MALDI results in the formation of an excessive number of weak complexes with the matrix. Broadband excitation in, or heating of, the trap could be used to break these complexes and provide cleaner peaks of molecular ions.
In another preferred embodiment, the trap/TOF pulse preparation stage is coupled to an ESI source with a modulating multipole ion guide. The trap in this embodiment is a linear two-dimensional segmented trap to allow a wide range of masses to be trapped, thereby substantially increasing the space charge capacity of the trap. The trap is connected to the TOF analyzer via an intermediate, differentially pumped stage. The ion beam is fully utilized, providing a 100% duty cycle. The drift component of ion velocity is essentially eliminated and ions are injected into the TOF parallel to the axis.
The invention further encompasses the use of dynamically trapped, collisionally cooled ion preparation as part of a tandem TOF system. The precursor ions are injected into a trap with the energy desired for collisional dissociation. In one embodiment, the injected pulsed beam is dynamically trapped, undergoes fragmentation in earlier collisions and the resulting collection of fragment and precursor ions are collisionally cooled in the trap. Thus, the event which promotes the increase in internal energy necessary for fragmentation (e.g., collisions with a surface or a background gas), the trapping electrodes, and the background neutral gas are in a common volume, and activation and dissociation occur simultaneous with trapping. In another embodiment of the invention, the precursor ions are activated (i.e., their internal kinetic energy is increased) by surface induced dissociation (SID). The fragment ions formed in the SID process sequentially bounce off the surface, are dynamically trapped by the RF field and then are slowly damped in gas collisions. In both aforementioned embodiments, the use of dynamic trapping to efficiently capture the ion pulse allows the gas pressure in the trapping volume, and thus the mass analyzer, to be reduced. Consequently there will be fewer scattering collisions during both ejection into, and flight through, the mass analyzer, thereby allowing higher resolution to be achieved.
The invention provides a significant improvement of beam characteristics in front of the second TOFMS, since kinetic energy is damped in gas collisions and ions are confined to the center of the trap. As a result, the resolution is improved, linear calibration is achieved, and operation of the analyzer is decoupled from the ionization source.
Briefly, a preferred embodiment of the system for tandem TOF instruments operates as follows:
(1) A pulsed ion beam is formed from a MALDI or ESI source.
(2) A precursor ion is selected. In this embodiment the method of selection is a linear TOF equipped with a timed ion selector. In order to increase resolution of selection, a reflecting system can be employed.
(3) The ions are decelerated to the desired injection energy. In this manner, there is control of the energy available for the activation event, trapping is ensured, and ions of different mass but identical velocity to the precursor are filtered prior to entering the fragmentor volume.
(4) The precursor ions are pulse injected into a fragmentor. The fragmentor could contain a surface for SID (such as a gold surface with a monolayer of an organic known to promote efficient conversion of translational kinetic energy to internal energy) and/or a relative high pressure (1xc3x9710xe2x88x922 to 1xc3x9710xe2x88x924 torr) neutral gas for CID. In either instance some fraction of the ion population rapidly (e.g., in 1 xcexcs to 1 ms) dissociates into fragment ions.
(5) The collection of activated precursor and fragment ions in the fragmentor is dynamically trapped and collisionally cooled for a fixed time frame as described above with respect to the TOF-only method. For tandem mass spectrometry applications, the trapping time is varied considering both the needs for collisional cooling and precursor dissociation kinetics.
(6) The contents of the fragmentor are extracted into the second TOFMS for fragment analysis using a uniform pulsed electric field.
(7) The fragments are mass analyzed by the second TOFMS as described above.
In one preferred embodiment, a folded geometry is employed, and the same mass analyzer is used for both MS1 and MS2. The beam is formed in a pulsed source and is passed through the orifice of an annular detector. The beam is reflected in an electrostatic mirror at a small angle to the TOF axis. Precursor ions are selected with high resolution in a timed ion selector and enter the collisonal cell, equipped with a segmented ion trap. Fragments are trapped, cooled, and ejected into the same TOF analyzer but in the reverse direction. After being reflected in the mirror the ions hit the detector. This embodiment of the invention provides an inexpensive and compact solution for TOFxe2x80x94TOF instruments.
The invention summarized above addresses the limitations in TOF analysis as previously described. In particular, confining the ions in a collisional environment between the source and the pulsing necessary for TOF analysis provides a period of relaxation such that excess internal energy may dissipate prior to analysis. This will xe2x80x9ccoolxe2x80x9d the internal temperature of the ions, lowering the rate of thermal decomposition, and thereby minimizing metastable fragmentation and the spectral noise associated with it. The combined use of a quadrupolar field, with collisional cooling, will result in the spatial localization of the low energy ions in the center of the electrode structure, thereby creating perfect initial conditions for extraction into the TOF analyzer. In addition, confining the ions to the center of the field will minimize the spatial spread of the extracted ions, largely eliminating the correlation between mass resolution and phase at the time of extraction. The segmented ring geometry provides an electrode geometry that can be used to create both a quadrupolar and an accelerating field. Additionally there should not be ion losses in the extraction phase. The present invention is presented as a general apparatus and method for preparing an ideal pulse for TOF analysis, and is easily adaptable to existing configurations of instruments.